Microscope and ghosting elimination method

ABSTRACT

A microscope includes a first imaging optical system that images sample transmitted light transmitted through a sample provided on a stage, and a second imaging optical system that images a part of the sample transmitted light branched from the first imaging optical system. Here, the second imaging optical system includes a light beam branching element that branches the part of the sample transmitted light from the first imaging optical system, and has a thickness of a predetermined threshold or more, an imaging element that images a phase difference image of the branched sample transmitted light, one or a plurality of optical elements that images an image of the phase difference image of the branched sample transmitted light on the imaging element, and a filter that shields a part of the branched sample transmitted light imaged on the imaging element.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application claims priority to Japanese Priority PatentApplication JP 2010-181760 filed in the Japan Patent Office on Aug. 16,2010, the entire contents of which are hereby incorporated by reference.

BACKGROUND

The present application relates to a microscope and a ghostingelimination method.

In the related art, a motor-driven microscope in which a condenser lens,a visual field diaphragm, an aperture diaphragm, the optical axisdirection driving mechanism for the objective lens of a sample stage, afilter, a dimming power supply with respect to a light source, and thelike are automatically adjusted in accordance with the switching of theobjective lens has been disclosed (For example, see Japanese UnexaminedPatent Application Publication No. 11-133311).

SUMMARY

However, in order to achieve an autofocus function with respect to themicroscope described in Japanese Unexamined Patent ApplicationPublication No. 11-133311, attaching an autofocus device using a phasedifference optical system that obtains a defocus position to be observedto the microscope has been considered. In such a case, a light beambranching element is provided on the optical axis of an imaging opticalsystem for imaging, on an imaging element, light transmitted through asample, so that a part of the light transmitted through the sample isguided to the phase difference optical system.

As a result of intensive studies made by the inventors, with respect tothe autofocus device using the phase difference optical system, it wasfound that ghosting caused by the light beam branching element affectsan image that is imaged by the imaging optical system and the phasedifference optical system.

The present application is to solve the above problem, and it isdesirable to provide a microscope having an autofocus function using aphase difference optical system, and provide a ghosting eliminationmethod in which ghosting caused by a light beam branching element iseliminated with respect to the microscope.

According to an embodiment, there is provided a microscope, including: afirst imaging optical system that images sample transmitted lighttransmitted through a sample provided on a stage; and a second imagingoptical system that images a part of the sample transmitted lightbranched from the first imaging optical system. Here, the second imagingoptical system may include a light beam branching element that branchesthe part of the sample transmitted light from the first imaging opticalsystem, and has a thickness of a predetermined threshold or more, animaging element that images a phase difference image of the branchedsample transmitted light, one or a plurality of optical elements thatimages an image of the phase difference image of the branched sampletransmitted light on the imaging element, and a filter that shields apart of the branched sample transmitted light focused on the imagingelement.

The sample transmitted light reflected by the light beam branchingelement may be imaged in the first imaging optical system, and the phasedifference image of the sample transmitted light transmitted through thelight beam branching element may be imaged in the second imaging opticalsystem.

The sample transmitted light transmitted through the light beambranching element may be imaged in the first imaging optical system, andthe phase difference image of the sample transmitted light reflected bythe light beam branching element may be imaged in the second imagingoptical system.

The filter may be a diaphragm in which a through hole set for allowing aluminous flux set which becomes the phase difference image to passtherethrough is provided, and the thickness of the light beam branchingelement may have a larger value than a feature value calculated based ona diameter of the through hole, a center distance between the throughholes of the through hole set, and a luminous flux diameter at theposition of the filter of the sample transmitted light.

The thickness of the light beam branching element may have a largervalue than a feature value calculated based on the following Inequality1, which is represented as

$\begin{matrix}{t > {k \times \frac{\left( {{\varphi \; a} + {\varphi \; b} + d} \right)}{2}}} & \left\lbrack {{Inequality}\mspace{14mu} 1} \right\rbrack\end{matrix}$

where t denotes the thickness of the light beam branching element, kdenotes a specific constant in an optical system, φa denotes a luminousflux diameter at a filter position of the sample transmitted light, φbdenotes the diameter of the through hole, and d denotes the distancebetween centers of the through holes of the through hole set.

According to another embodiment, there is provided a ghostingelimination method, including: branching, by a light beam branchingelement having a thickness of a predetermined threshold or more, a partof sample transmitted light transmitted through a sample provided on astage; and shielding, by an imaging element for imaging a phasedifference image of the branched sample transmitted light and a filterprovided between the imaging element and the light beam branchingelement, ghosting light caused by a corresponding light beam branchingelement from the part of the sample transmitted light branched by thelight beam branching element.

As described above, it is possible to eliminate ghosting caused by alight beam branching element with respect to a microscope having anautofocus function using a phase difference optical system.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a configuration of a microscope according toa first embodiment;

FIG. 2 is a block diagram showing a configuration of an integrationcontrol unit according to a first embodiment;

FIG. 3 is a diagram showing a light beam branching element;

FIG. 4 is a diagram showing an example of a defocus quantity detectionunit according to a first embodiment;

FIG. 5 is a graph showing a relationship between a surface transmittanceof a light beam branching element and an SN ratio of ghosting;

FIG. 6 is a graph showing a relationship between a thickness of a lightbeam branching element and an amount of deflection;

FIG. 7 is a diagram showing an example of a two eye lens filteraccording to a first embodiment;

FIG. 8 is a graph showing a relationship between a wavefront aberrationand a thickness of an optical branching element; and

FIG. 9 is a schematic diagram showing an example of a defocus quantitydetection unit according to a second embodiment.

DETAILED DESCRIPTION

Embodiments of the present application will be described below in detailwith reference to the drawings.

Further, the description will be made in the following order:

(1) First Embodiment

(1-1) Configuration of microscope

(1-2) Light beam branching element

(1-3) configuration of defocus quantity detection unit

(2) Second Embodiment

First Embodiment

<Configuration of Microscope>

A configuration of a microscope 1 according to a first embodiment willbe described with reference to FIG. 1. FIG. 1 is a diagram showing aconfiguration of a microscope 1 according to the present embodiment.

[Entire Configuration]

As shown in FIG. 1, the microscope 1 according to the present embodimentincludes a thumbnail image imaging unit 10 for imaging an image(hereinafter, referred to as “thumbnail image”) of the entirepreparation PRT in which a biological sample SPL is embedded, and amagnified image imaging unit 20 for imaging an image (hereinafter,referred to as “magnified image”) in which the biological sample SPL ismagnified at a predetermined magnification ratio. In addition, in themagnified image imaging unit 20, a defocus quantity detection unit 30for detecting a defocus quantity of an illumination visual fielddiaphragm existing within the magnified image imaging unit 20 isprovided.

The preparation PRT is obtained by fixing, on a slide glass, thebiological sample SPL including tissue sections such as connectivetissue such as blood, epithelial tissue, or both of these organizations,or smear cells using a predetermined fixing means. The tissue sectionsor the smear cells are subjected to various types of staining, asnecessary. This staining includes general staining represented by HE(Hematoxylin and Eosin) staining, Giemsa staining, and Papanicolaoustaining, and fluorescent staining such as FISH (Fluorescence In-SituHybridization), an enzyme antibody method, or the like.

In addition, a label in which supplementary information (for example,name of the person who collects sample, collection date, and type ofstaining, and the like) for specifying a corresponding biological sampleSPL is described is attached to the preparation PRT.

In the microscope 1 according to the present embodiment, a stage 40 inwhich the above described preparation PRT is placed, and a stage drivingmechanism 41 for moving the stage 40 in a variety of directions areprovided. By the stage driving mechanism 41, the stage 40 is freelymoved in a direction (Z-axis direction) perpendicular to a direction(X-axis and Y-axis direction) parallel to a stage surface.

In addition, in the magnified image imaging unit 20, a condenser lensdriving mechanism 42 as an example of a focus adjustment unit for anillumination visual field diaphragm is provided.

<Thumbnail Image Imaging Unit>

As shown in FIG. 1, the thumbnail image imaging unit 10 mainly includesa light source 11, an objective lens 12, and an imaging element 13.

The light source 11 is provided on a surface opposite to a surface onwhich the preparation of the stage 40 is disposed. The light source 11may perform irradiation by switching light (hereinafter, referred to asbright visual field illumination light, or simply referred toillumination light) for illuminating the biological sample SPL havingbeen subjected to the general staining, and light (hereinafter, referredto as dark visual field illumination light) for illuminating thebiological sample SPL having been subjected to special staining. Inaddition, the light source 11 may perform irradiation with respect toonly one of the bright visual field illumination light and the darkvisual field illumination light. In this case, as the light source 11,two types of light sources such as a light source for irradiating thebright visual field illumination light and a light source forirradiating the dark visual field illumination light are provided.

In addition, in the thumbnail image imaging unit 10, a label lightsource (not shown) that irradiates light for imaging the supplementaryinformation described on the label attached to the preparation PRT maynot be separately provided.

The objective lens 12 with a predetermined magnification is arranged onthe preparation disposition surface side of the stage 40 using, as theoptical axis SRA, a normal line of a reference position of the thumbnailimage imaging unit 10 in the preparation disposition surface.Transmitted light transmitted through the preparation PRT arranged onthe stage 40 is condensed by the objective lens 12, and is imaged on theimaging element 13 provided behind (that is, advancing direction of theillumination light) the objective lens 12.

On the imaging element 13, light (in other words, transmitted lighttransmitted through the entire preparation PRT) of an imaging rangeincluding the entire preparation PRT placed in the preparationdisposition surface of the stage 40 is imaged. An image imaged on theimaging element 13 is a thumbnail image being a microscope imageobtained by imaging the entire preparation PRT.

<Magnified Image Imaging Unit>

As shown in FIG. 1, the magnified image imaging unit 20 mainly includesa light source 21, a condenser lens 22, an objective lens 23, and animaging element 24. In addition, an illumination visual field diaphragm(not shown) is provided in the magnified image imaging unit 20.

The light source 21 irradiates bright visual field illumination light,and is provided on a surface opposite to a surface on which thepreparation of the stage 40 is disposed. In addition, a light source(not shown) that irradiates dark visual field illumination light isprovided in different position (for example, preparation dispositionsurface side) from that of the light source 21.

The condenser lens 22 is a lens that condenses the bright visual fieldillumination light irradiated from the light source 21 or the darkvisual field illumination light irradiated from a light source for darkvisual field illumination, and guides the condensed light to thepreparation PRT on the stage 40. The condenser lens 22 is arrangedbetween the light source 21 and the stage 40 using, as the optical axisERA, a normal line of a reference position of the magnified imageimaging unit 20 in the preparation disposition surface. In addition, thecondenser lens driving mechanism 42 may drive the condenser lens 22 in adirection of the optical axis ERA. The condenser lens 22 may change theposition on the optical axis ERA by the condenser lens driving mechanism42.

The objective lens 23 of the predetermined magnification is arranged inthe preparation disposition surface side of the stage 40 using, as theoptical axis ERA, the normal line of the reference position of themagnified image imaging unit 20 in the preparation disposition surface.In the magnified image imaging unit 20, the biological sample SPL ismagnified at a variety of magnification ratios by appropriately changingthe objective lens 23, and the magnified sample is imaged. Thetransmitted light transmitted through the preparation PRT arranged onthe stage 40 is condensed by the objective lens 23, and is imaged on theimaging element 24 provided behind (that is, advancing direction ofillumination light) the objective lens 23.

In addition, on the optical axis ERA between the objective lens 23 as anexample of a first imaging optical system, and the imaging element 24, alight beam branching element 31 is provided, and a part of thetransmitted light transmitted through the objective lens 23 is guided toa defocus quantity detection unit 30, which will be described later.

In the imaging element 24, an image of an imaging range having apredetermined width and height is imaged on the preparation dispositionsurface of the stage 40 depending on a pixel size of the imaging element24 and a magnification ratio of the objective lens 23. In addition,since a part of the biological sample SPL is magnified by the objectivelens 23, the above described imaging range is a satisfactorily narrowrange, compared with the imaging range of the imaging element 13.

Here, as shown in FIG. 1, the thumbnail image imaging unit 10 and themagnified image imaging unit 20 are arranged in such a manner that theoptical axes SRA and ERA of the normal line of the reference position ofeach of the thumbnail image imaging unit 10 and the magnified imageimaging unit 20 are separated from each other by a distance D in aY-axis direction. The distance D is set to a short distance forminiaturization while a lens barrel (not shown) holding the objectivelens 23 of the magnified image imaging unit 20 does not appear in theimaging range of the imaging element 13.

<Defocus Quantity Detection Unit>

The defocus quantity detection unit 30 as an example of a second imagingoptical system mainly includes a light beam branching element 31, acondenser lens 32, a two eye lens filter 33, a two eye lens 34, and animaging element 35, as shown in FIG. 1.

As described above, the light beam branching element 31 is provided onthe optical axis ERA between the objective lens 23 of the magnifiedimage imaging unit 20 and the imaging element 24, so that a part ofsample transmitted light (light transmitted through the sample)transmitted through the objective lens 23 is reflected. In other words,by the light beam branching element 31, the sample transmitted lighttransmitted through the objective lens 23 is branched to transmittedlight advancing toward the imaging element 24 and reflected lightadvancing toward the condenser lens 32 within the defocus quantitydetection unit 30, which will be described later.

According to the present embodiment, in an advancing direction side ofthe reflected light branched by the light beam branching element 31, thecondenser lens 32 is provided. The condenser lens 32 condenses thereflected light branched by the light beam branching element 31, andguides the condensed light to the two eye lens 34 provided behind(advancing direction side of reflected light) the condenser lens 32.

The two eye lens filter 33 is a filter that is provided between thecondenser lens 32 and the two eye lens 34, which will be describedlater, and shields a part of the reflected light (reflected light ofsample transmitted light) imaged on the imaging element 35 providedwithin the defocus quantity detection unit 30. The reflected lighttransmitted through the two eye filter 33 is guided to the two eye lens34 provided behind the two eye lens filter 33.

The two eye lens 34 splits a luminous flux introduced by the condenserlens 32 into two luminous fluxes. The split luminous flux forms a set ofobject images on a imaging surface of the imaging element 35 providedbehind (advancing direction side of reflected light) the two eye lens34.

On the imaging element 35, each of light transmitted through the two eyelens 34 is imaged. As a result, on an imaging surface of the imagingelement 35, the set of object images is formed. Since luminous fluxes ofa variety of directions emitted from the condenser lens 32 are madeincident on the two eye lens 34, a phase difference exists between theformed set of object images. Hereinafter, the set of object images isreferred to as a phase difference image. The defocus quantity detectionunit 30 according to the present embodiment detects a defocus quantityof an illumination visual field diaphragm existing within the magnifiedimage imaging unit 20, using the phase difference.

Further, in the described above, a configuration in which the condenserlens as a phase difference AF optical system within the defocus quantitydetection unit 30, the two eye lens filter, the two eye lens, and theimaging element are provided is shown, however, the configuration is notlimited to the example. Another optical system may be used as long asthe other optical system can realize the same function as that of thephase difference AF optical system, such as using a field lens and aseparator lens instead of the condenser lens and the two eye lens.

In addition, the imaging elements which are provided on the thumbnailimage imaging unit 10, the magnified image imaging unit 20 and thedefocus quantity detection unit 30 respectively, may be an onedimensional imaging element or a two dimensional element.

Further, the defocus quantity detection unit 30 will be described indetail below again.

<Control Unit>

As shown in FIG. 1, a control unit for controlling various positions ofthe microscope is connected to the microscope 1 according to the presentembodiment. Specifically, an illumination control unit 51 forcontrolling a variety of light sources of the microscope 1 whichincludes a light source 11 and a light source 21 is connected to themicroscope 1 according to the present embodiment, and a stage drivingcontrol unit 52 for controlling the stage driving mechanism 41 isconnected to the stage driving mechanism 41. In addition, a condenserlens driving control unit 53 for performing position control of thecondenser lens 22 is connected to the condenser lens 22. Further, aphase difference image imaging control unit 54 is connected to theimaging element 35 for imaging the phase difference image, and athumbnail image imaging control unit 55 is connected to the imagingelement 13 for imaging a thumbnail image. In addition, a magnified imageimaging control unit 56 is connected to the imaging element 24 forimaging a magnified image of the biological sample SPL. These controlunits are connected with respect to a position for performing controlvia a variety of data communication channels.

In addition, in the microscope 1 according to the present embodiment, acontrol unit (hereinafter, referred to as integrated control unit 50)for controlling the entire microscope is separately provided, andconnected to the above described control units via the variety of datacommunication channels.

The above described control unit is realized by CPU (Central ProcessingUnit), GPU (Graphics Processing Unit), ROM (Read Only Memory), RAM(Random Access Memory), a storage device, a communication device, anarithmetic circuit, and the like.

Hereinafter, function of the above described control unit will bebriefly described.

<Illumination Control Unit>

An illumination control unit 51 is a processing unit for controlling avariety of light sources of the microscope 1 according to the presentembodiment. The illumination control unit 51 performs irradiationcontrol of a corresponding light source based on information indicatingan acquired illumination method when information indicating anillumination method of the biological sample SPL is output from theintegrated control unit 50.

For example, a case in which the illumination control unit 51 controlsthe light source 11 provided in the thumbnail image imaging unit 10 willbe herein noted. In such a case, with reference to informationindicating the illumination method, the illumination control unit 51determines which one of a mode (hereinafter, referred to as brightvisual field mode) which is necessary for acquiring a bright visualfield image, and a mode (hereinafter, referred to as dark visual fieldmode) which is necessary for acquiring a dark visual field image isperformed. Thereafter, the illumination control unit 51 sets parametersdepending on each mode with respect to the light source 11, andirradiates illumination light applied to each mode from the light source11. Thus, the illumination light irradiated from the light source 11 isirradiated to the entire biological sample SPL via an opening of thestage 40. Further, as examples of the parameters set by the illuminationcontrol unit 51, intensity of the illumination light, selection in typeof the light source, and the like may be given.

In addition, a case in which the illumination control unit 51 controlsthe light source 21 provided in the magnified image imaging unit 20 willbe herein noted. In such a case, with reference to informationindicating the illumination method, the illumination control unit 51determines which one of the bright visual field mode and the dark visualfield mode is performed. Thereafter, the illumination control unit 51sets parameters depending on each mode with respect to the light source21, and irradiates illumination light applied to each mode from thelight source 21. Thus, the illumination light irradiated from the lightsource 21 is irradiated to the entire biological sample SPL via theopening of the stage 40. Further, as examples of the parameters set bythe illumination control unit 51, intensity of the illumination light,selection in the type of the light source, and the like may be given.

Further, as irradiation light in the bright visual field mode, visiblelight may be used. In addition, as irradiation light in the dark visualfield mode, light including a wavelength which can excite a fluorescentmarker used in the special staining may be used. In addition, in thedark visual field mode, a background portion with respect to thefluorescent marker is cut out.

<Stage Driving Control Unit>

The stage driving control unit 52 is a processing unit that controls thestage driving mechanism 41 for driving the stage provided in themicroscope 1 according to the present embodiment. The stage drivingcontrol unit 52 controls the stage driving mechanism 41 based oninformation indicating an acquired imaging method when informationindicating an imaging method of the biological sample SPL is output fromthe integrated control unit 50.

For example, a case in which a thumbnail image is imaged by themicroscope 1 according to the present embodiment will be noted herein.When information indicating that the thumbnail image of the biologicalsample SPL is imaged is output from the integrated control unit 50, thestage driving control unit 52 moves the stage 40 in a stage surfacedirection (X-Y axis direction) so that the entire preparation PRT iswithin the imaging range of the imaging element 13. In addition, thestage driving control unit 52 moves the stage 40 in a Z-axis directionso that focal point of the objective lens 12 matches the entirepreparation PRT.

In addition, a case in which a magnified image is imaged by themicroscope 1 according to the present embodiment will be herein noted.When information indicating that the magnified image of the biologicalsample SPL is imaged is output from the integrated control unit 50, thestage driving control unit 52 drives and controls the stage drivingmechanism 41, and moves the stage 40 in the stage surface direction sothat the biological sample SPL is positioned between the light source 11and the objective lens 12 and between the condenser lens 22 and theobjective lens 23.

In addition, the stage driving control unit 52 moves the stage 40 in thestage surface direction (X-Y axis direction) so that a predeterminedportion of the biological sample is positioned in the imaging rangeimaged by the imaging element 24.

Further, the stage driving control unit 52 moves the stage 40 in adirection (Z-axis direction, and depth direction of tissue sections)perpendicular to the stage surface so that the position of thebiological sample SPL positioned within a predetermined shooting rangematches the focal point of the objective lens 23 by driving andcontrolling the stage driving mechanism 41.

<Condenser Lens Driving Control Unit>

The condenser lens driving control unit 53 is a processing unit thatcontrols the condenser lens driving mechanism 42 for driving thecondenser lens 22 provided in the magnified image imaging unit 20 of themicroscope 1 according to the present embodiment. When informationrelating to the defocus quantity of the illumination visual fielddiaphragm is output from the integrated control unit 50, the condenserlens driving control unit 53 controls the condenser lens drivingmechanism 42 based on acquired information relating to the defocusquantity.

As described below, when the illumination visual field diaphragmprovided within the magnified image imaging unit 20 is not properlyimaged, a generated contrast of the magnified image is degraded. Inorder to prevent the degradation of the contrast, in integrated controlunit 50 which will be described later, a specification processing isperformed with respect to the defocus quantity of the illuminationvisual field diaphragm based on a phase difference image generated bythe defocus quantity detection unit 30, in the microscope 1 according tothe present embodiment. The integrated control unit 50 outputsinformation indicating the specified defocus quantity of theillumination visual field diaphragm to the condenser lens drivingcontrol unit 53, and changes the position of the condenser lens 22 sothat the illumination visual field diaphragm is imaged.

So that the illumination visual field diaphragm is imaged by performingdriving control of the condenser lens driving mechanism 42, thecondenser lens driving control unit 53 corrects the position (positionon the optical axis ERA) of the condenser lens 22.

<Phase Difference Image Imaging Control Unit>

The phase difference image imaging control unit 54 is a processing unitthat controls the imaging element 35 provided in the defocus quantitydetection unit 30. The phase difference image imaging control unit 54sets parameters according to the bright visual field mode or the darkvisual field mode in the imaging element 35. In addition, when acquiringoutput signals, which are output from the imaging element 35, equivalentto an image imaged on the imaging surface of the imaging element 35, thephase difference image imaging control unit 54 uses the acquired outputsignals as output signals equivalent to the phase difference image. Whenacquiring the output signals equivalent to the phase difference image,the phase difference image imaging control unit 54 outputs dataequivalent to the acquired signals to the integrated control unit 50.Further, as examples of the parameters set by the phase difference imageimaging control unit 54, a start timing and a termination timing ofexposure (in other words, exposure time), and the like may be given.

<Thumbnail Image Imaging Control Unit>

The thumbnail image imaging control unit 55 is a processing unit thatcontrols the imaging element 13 provided in the thumbnail image imagingunit 10. The thumbnail image imaging control unit 55 sets parametersaccording to the bright visual field mode or the dark visual field modein the imaging element 13. In addition, when acquiring output signalscorresponding to an image imaged on the imaging surface of the imagingelement 13 which is output from the imaging element 13, the thumbnailimage imaging control unit 55 uses the acquired output signals as outputsignals corresponding to the thumbnail image. When acquiring the outputsignals corresponding to the thumbnail image, the thumbnail imageimaging control unit 55 outputs data corresponding to the acquiredsignals to the integrated control unit 50. Further, as examples of theparameters set by the thumbnail image imaging control unit 55, a starttiming and a termination timing of exposure (in other words, exposuretime), and the like may be given.

<Magnified Image Imaging Control Unit>

The magnified image imaging control unit 56 is a processing unit thatcontrols the imaging element 24 provided in the magnified image imagingunit 20. The magnified image imaging control unit 56 sets parametersaccording to the bright visual field mode or the dark visual field modein the imaging element 24. In addition, when acquiring output signalscorresponding to an image imaged on the imaging surface of the imagingelement 24 which is output from the imaging element 24, the magnifiedimage imaging control unit 56 uses the acquired output signals as outputsignals corresponding to a magnified image. When acquiring the outputsignals corresponding to the magnified image, the magnified imageimaging control unit 56 outputs data corresponding to the acquiredsignals to the integrated control unit 50. Further, as examples of theparameters set by the magnified image imaging control unit 56, a starttiming and a termination timing of exposure (in other words, exposuretime), and the like may be given.

The storage unit 57 is an example of a storage device included in themicroscope 1 according to the present embodiment. In the storage unit57, various setting information for controlling the microscope 1according to the present embodiment, various databases, or a look-uptable, or the like is stored. In addition, in the storage unit 57, avariety of historical information such as an imaging history of thesample in the microscope 1 may be recorded. Further, in the storage unit57, various parameters have to be saved when performing certainprocessing by the microscope 1 (particularly, integrated control unit50) according to the present embodiment, a progress of the process,various databases or programs, and the like are appropriately recorded.

In the storage unit 57, it is possible for the respective processingunits included in the microscope 1 to freely perform reading andwriting.

<Integrated Control Unit>

The integrated control unit 50 is a processing unit that controls theentire microscope including the above described various control units.

The integrated control unit 50 acquires data relating to the phasedifference image imaged by the microscope 1, and calculates a defocusquantity of the illumination visual field diaphragm and the amount ofchange in the thickness of a slide glass, based on the phase differenceimage data. The integrated control unit 50 executes imaging of theoptical system present within the magnified image imaging unit 20 of themicroscope 1 using the defocus quantity and the amount of change in thethickness of the slide glass, so that it is possible to further improveimaging precision of the obtained magnified image.

In addition, the integrated control unit 50 acquires, from themicroscope 1, microscope image data relating to the thumbnail image andthe magnified image which are imaged by the microscope 1, and developsthis data, or executes a predetermined digital processing. Thereafter,the integrated control unit 50 uploads the microscope image dataobtained from the thumbnail image and the magnified image to an imagemanagement server via a network such as the Internet, a dedicated line,and the like. Thus, the microscope image of the sample imaged by themicroscope 1 is able to be browsed by a client device connected to thenetwork.

Hereinafter, with reference to FIG. 2, a configuration of the integratedcontrol unit 50 according to the present embodiment will be described indetail. FIG. 2 is a block diagram showing a configuration of theintegrated control unit 50 according to the present embodiment.

As shown in FIG. 2, the integrated control unit 50 according to thepresent embodiment mainly includes an integrated driving control unit501, an image acquisition unit 503, an image processing unit 505, afeature value calculation unit 507, a microscope image output unit 509,and a communication control unit 511.

The integrated driving control unit 501 is realized by, for example,CPU, ROM, RAM, or the like. The integrated driving control unit 501 is adriving control unit that integrally controls the control unit (theillumination control unit 51, the stage driving control unit 52, thecondenser lens driving control unit 53, and the phase difference imageimaging control unit 54, the thumbnail image imaging control unit 55,and the magnified image imaging control unit 56) for controlling eachpart of the microscope 1. The integrated driving control unit 501 sets avariety of information (for example, various setting parameters, and thelike) with respect to each part of the microscope 1, or acquires avariety of information from each part of the microscope 1. Theintegrated driving control unit 501 may output the variety ofinformation acquired from each part of the microscope 1 to the featurevalue calculation unit 507, which will be described later.

The image acquisition unit 503 is realized by, for example, CPU, ROM,RAM, a communication device, or the like. The image acquisition unit 503acquires data corresponding to the thumbnail image imaged by thethumbnail image imaging unit 10, data corresponding to the magnifiedimage imaged by the magnified image imaging unit 20, and datacorresponding to the phase difference image imaged by the defocusquantity detection unit 30 through each imaging control unit.

When acquiring the image data through each imaging control unit, theimage acquisition unit 503 outputs the acquired image data to the imagecontrol unit 225, which will be described later.

Further, the image acquisition unit 503 may associate the acquired imagedata (the microscope image data) with information concerning anacquisition date, and the like, and store the associated information inthe storage unit 57, and the like.

The image processing unit 505 is realized by, for example, CPU, GPU,ROM, RAM, and the like. The image processing unit 505 executes apredetermined image process on the microscope image output from theimage acquisition unit 503.

Specifically, when acquiring the phase difference image data, thethumbnail image data, and the magnified image data (more specifically,RAW data of these images) which are output from the image acquisitionunit 503, the image processing unit 505 performs a developing process ofthe RAW data. In addition, the image processing unit 505 executes aprocess (stitching process) in which a plurality of images constitutingthese images is connected together, while performing the developingprocess of the image data.

In addition, the image processing unit 505 is able to execute aconversion process (transcoding) of acquired digital image data, ifnecessary. As the conversion process of the digital image, a process inwhich the digital image is compressed to generate a JPEG image, or aprocess in which the data compressed into the JPEG image is convertedinto a compressed image of a different type (for example, GIF format,and the like) may be given. In addition, in the conversion process ofthe digital image, a re-compressing process in which the compressedimage data is decompressed once, and then is subjected to a process suchas edge enhancement, and the like, or a process of changing thecompression ratio of the compressed image may be included.

When the above described image process is executed with respect to thephase difference image data, the image processing unit 505 outputs thephase difference image data obtained after executing the image processto the feature value calculation unit 507, which will be describedlater. In addition, when the above described image process is executedwith respect to the thumbnail image data and the magnified image data,the image processing unit 505 outputs the microscope image obtained fromthese images and various metadata representing the microscope image tothe microscope image output unit 509, which will be described later.

The feature value calculation unit 507 is realized by, for example, CPU,GPU, ROM, RAM, or the like. The feature value calculation unit 507acquires data concerning the phase difference image imaged by themicroscope 1, and calculates a defocus quantity of the sample placed inthe stage of the microscope 1 based on the phase difference image data.In addition, the feature value calculation unit 507 is able to calculatethe defocus quantity of the illumination visual field diaphragm, and theamount of change in the thickness of the slide glass based on the phasedifference image data. The integrated control unit 50 executes imagingof the optical system present within the magnified image imaging unit 20of the microscope 1 using the defocus quantity and the amount of changein the thickness of the slide glass, so that it is possible to furtherimprove imaging precision of the obtained magnified image.

Various feature values described above that are calculated by thefeature value calculation unit 507 are output to the integrated drivingcontrol unit 501.

The microscope image output unit 509 is realized by, for example, CPU,ROM, RAM, or the like. The microscope image output unit 509 outputs, tothe image management server through the communication control unit 511which will be described later, a variety of information such as themicroscope image output from the image processing unit 505, metadataassociated with the microscope image, and the like. Thus, the microscopeimage (digital microscope image) of the sample imaged by the microscope1 is managed by the image management server.

The communication control unit 511 is realized by, for example, CPU,ROM, RAM, a communication device, or the like. The communication controlunit 511 performs control of the communication performed through anetwork such as the Internet, a dedicated line, and the like between theintegrated control unit 50 and the image management server providedexternally to microscope 1.

As above, an example of the functions of the integrated control unit 50according to the present embodiment has been shown. The above describedcomponents may be configured using members or circuits for generalpurpose, or configured by hardware specialized for the function of eachof the components. In addition, the functions of each of the componentsare entirely performed by CPU, and the like. Accordingly, depending onlevels of technologies when the present embodiment is executed, it ispossible to appropriately change the configuration to be used.

Further, it is possible to prepare a computer program for realizing eachfunction of the integrated control unit according to the presentembodiment or of other control units, and to implement the preparedcomputer program in a personal computer, and the like. In addition, itis possible to provide a computer-readable recording medium in which theabove described computer program is stored. The recording medium is, forexample, a magnetic disk, an optical disc, a magneto-optical disc, aflash memory, and the like. In addition, the computer program may bedelivered over, for example, the network without using the recordingmedium.

As above, the entire configuration of the microscope 1 according to thepresent embodiment has been described in detail with reference to FIGS.1 to 2.

<Light Beam Branching Element>

Next, before describing the defocus quantity detection unit 30 accordingto the present embodiment in detail, studies for the light beambranching element conducted by the present inventors will be describedin detail with reference to FIG. 3. FIG. 3 is a diagram showing thelight beam branching element.

As shown in FIG. 3, light made incident on the light beam branchingelement is branched to reflected light R1 reflected from a front surfaceof the light beam branching element, and transmitted light T1T2transmitted through the front surface and the rear surface of the lightbeam branching element. However, the light made incident on the lightbeam branching element is multiplex-reflected within the light beambranching element to thereby become reflected light called ghostinglight or transmitted light, other than the reflected light R1 and thetransmitted light T1T2.

For example, the light made incident on the light beam branching elementis reflected from the rear surface of the light beam branching element,and further escaped from the front surface of the light beam branchingelement (light beam shown in T1R2T1 of FIG. 3) to thereby becomeghosting light of the reflected light R1. Similarly, the light madeincident on the light beam branching element is reflected from the rearsurface and the front surface of the light beam branching element, andescaped from the rear surface of the light beam branching element (lightbeam shown in T1R2R1T2 of FIG. 3) to thereby become ghosting light ofthe transmitted light T1T2. The reflected light or the transmitted lightand the ghosting light corresponding to this light are separated fromeach other by an interval Δ as shown in FIG. 3, and image blurring orthe like is caused by the ghosting light.

The separation distance Δ between the transmitted light or the reflectedlight and the ghosting light is changed depending on a thickness t ofthe light beam branching element, and when a reflective index n of thelight beam branching element is uniform, Δ is reduced along with areduction in the thickness t. Due to this, in the related art, it ispossible to match the ghosting light with the transmitted light or thereflected light as much as possible by reducing the thickness t.

In addition, as described above, since the ghosting light is generatedby reflection within the light beam branching element, the light beambranching element is subjected to an AR coating, and the like in therelated art, and thereby generation of the ghosting light in thetransmitted side or the reflected side is suppressed.

Here, as is apparent from FIG. 1, in the microscope 1 according to thepresent embodiment, both the transmitted light and the reflected lightof the light beam branching element are used in various processesperformed within the microscope 1, so that it is necessary thatgeneration of the ghosting light in both the transmitted side and thereflected side is suppressed.

However, even though the coating is executed on the light beam branchingelement, it is difficult for the generation of the ghosting light inboth the transmitted side and the reflected side to be suppressed. Inaddition, studies for using a pellicle mirror as the light beambranching element have been conducted by the present inventors, however,it was found that there are problems such as temperaturecharacteristics, disturbance of wave front due to deflection of themirror itself, and the like.

Therefore, the present inventors have conducted extensive studies for amethod of capable of removing ghosting due to the light beam branchingelement using luminous fluxes of both of the transmitted side and thereflected side in the microscope including a phase difference autofocusoptical system. As a result, as described below, the microscope 1according to the present embodiment is obtained.

<Configuration of Defocus Quantity Detection Unit>

Hereinafter, a phase difference autofocus (AF) optical system(hereinafter, simply referred to as phase difference optical system)included in the defocus quantity detection unit 30 according to thepresent embodiment will be described in detail with reference to FIGS. 4to 7.

As shown in FIG. 4, the phase difference optical system included in thedefocus quantity detection unit 30 according to the present embodimentincludes a light beam branching element 31 for branching a part of light(sample transmitted light) transmitted through a sample SPL, a condenserlens 32 for condensing the sample transmitted light branched by thelight beam branching element 31, a two eye lens filter 33, a two eyelens 34, and an imaging element 35.

Here, the light beam branching element 31 according to the presentembodiment is provided within an imaging optical system that includesthe condenser lens 23 for condensing the light (sample transmittedlight) transmitted through the sample SPL, and the imaging element 24 onwhich the sample transmitted light condensed by the condenser lens 23 isimaged. In addition, since the light beam branching element 31 accordingto the present embodiment eliminates the above described ghosting light,the light beam branching element 31 has a thickness more than apredetermined threshold value.

As described above, a separation distance Δ between the transmittedlight or the reflected light and the ghosting light is proportional tothe thickness t of the light beam branching element. Thus, in order toseparate the ghosting light from the transmitted light and the reflectedlight used in the microscope 1 as much as possible, a thickness of thelight beam branching element 31 is to be thicker than that of the lightbeam branching element in the related art. The light beam branchingelement 31 has a thickness more than a predetermined threshold value, sothat the ghosting light generated in the light beam branching element 31is separated significantly from the sample transmitted light (surfacereflected light) reflected by the light beam branching element 31. As aresult, it is possible to shield the ghosting light separatedsignificantly from the surface reflected light by the two eye lensfilter 33, which will be described later, so that it is possible toprevent the ghosting light from being imaged on the imaging element 35.

Here, by increasing the thickness of the light beam branching element31, an amount of deflection of the light beam branching element ischanged. FIG. 5 is a graph showing a result in which a flat plate usingBK7 as a glass material is fixed in such a manner that free ends of theflat plate are fixed, and the amount of deflection of the glass materialis measured. As is apparent from FIG. 5, it is found that the amount ofdeflection generated in the glass material is rapidly increased alongwith a reduction in the thickness of the glass material. In addition, inFIG. 5, the case of using the BK7 as the glass material is shown,however, a behavior such that the amount of deflection is increasedalong with the reduction in the thickness is common even using otherglass materials.

In the case of manufacturing the light beam branching element using theglass material, the reflected surface of the light beam branchingelement functions as a curvature reflection mirror different from anoriginal plane reflection mirror due to the deflection of the light beambranching element. Therefore, such as the light beam branching element31 according to the present embodiment, the thickness of the light beambranching element is increased reversely to the related art, so that itis possible to achieve separation of the ghosting light, and to preventthe deflection of the light beam branching element. Here, to preventeffect due to the above described deflection, in the light beambranching element 31 according to the present embodiment, a platethickness of the light beam branching element 31 is preferably increasedso that the deflection is negligible.

Further, the glass material used when manufacturing the light beambranching element is preferably selected in consideration of temperaturecharacteristics of the glass material, and the like. For example, sincechange in the plate thickness of the light beam branching element due tothermal expansion is proportional to a thermal expansion coefficient ofthe glass material, synthetic quartz having the thermal expansioncoefficient of about 8% is used in comparison with the thermal expansioncoefficient of the BK7, and the like, so that it is possible to suppresseffects relating to the thermal expansion, and to further suppresseffects caused by the change in the thickness.

In addition, as described above, the light beam branched by the lightbeam branching element 31 is used to acquire focus information (defocusinformation) by the defocus quantity detection unit 30 (phase differenceoptical system). Thus, so that even a defocused light beam is able to bedetected, the light beam branching element 31 preferably has a planesize which does not have light beam vignetting in a desired defocusrange.

As described above, in the microscope 1 according to the presentembodiment, the ghosting light is shielded by the two eye lens filterprovided within the phase difference optical system, so that it ispreferable that a desired amount of light is guided to the phasedifference optical system, and then effects of the ghosting lightgenerated in the imaging element 24 of the imaging optical system(magnified image imaging unit 20) are made small to a minimum.

For example, in a case in which the imaging element 24 provided in theimaging optical system is an imaging element with 12 bit gradation, itis preferable that the ratio SNR of the imaging element 24 to an amountof the ghosting light is at least 20 log(2¹²/1)=72.24 dB.

In addition, it is assumed that the front surface reflectance andtransmittance of the light beam branching element are R1 and T1,respectively, and the rear surface reflectance and transmittance are R2and T2. In this case, the ratio of the reflected side to the amount ofthe ghosting light is represented as 20 log(R1/(T1R2T1)), and the ratioof the transmitted side to the amount of the ghosting light isrepresented as 20 log(1/(R1R2)).

Thus, for example, an AR (Anti-Reflective) coating is performed on arear surface, and the amount of the ghosting light in a case in whichT2=0.98 and R2=0.02 is calculated to be shown in FIG. 6. In FIG. 6, sothat effect of the ghosting light is approximately the noise level ofthe imaging element 24 in the imaging element 24, it is preferable tosatisfy T1>0.988 and R1<0.012 when the imaging element 24 is provided onthe transmitted light side of the light beam branching element 31 asshown in FIG. 4.

In addition, according to a second embodiment, which will be describedlater, when the imaging element 24 is provided on the reflected lightside of the light beam branching element 31, it is preferable to satisfyT1<0.11 and R1>0.89.

Further, as for the coating performed on the light beam branchingelement 31, it is preferable to satisfy the above described conditionsover the whole range of wavelengths used in the microscope 1, that is, avisible region. In addition, in a case in which even an ultraviolet (UV)wavelength region and an infrared (IR) wavelength range are used in themicroscope 1, it is preferable that performance of the coating even inthese wavelength regions satisfies the above described conditions.

In addition, as for the coating performed on the light beam branchingelement 31, it is preferable to satisfy the above described conditionsover the whole angle of light beam which is made incident. For example,such as the microscope 1 according to the present embodiment shown inFIG. 4, when the light beam branching element 31 is installed to beinclined to the main light beam by 45°, it is preferable to satisfy theabove described conditions in a range including an off-axis light beamwith angle 45° as the center.

Further, using the front surface reflected light in the light beambranching element 31, effects due to a variety of plate thicknesses areable to be negligible. Thus, it is preferable that the front surfacereflected light is guided to the imaging element 24 installed on thereflected light side or the phase difference optical system.

Next, the thickness of the light beam branching element 31 will befurther described in consideration of a relationship between thethickness of the light beam branching element 31 and the two eye lensfilter 33 with reference to FIG. 7. FIG. 7 is a diagram showing anexample of the two eye lens filter 33 according to the presentembodiment.

As shown in FIG. 7, the two eye lens filter 33 according to the presentembodiment is a diaphragm in which a set of through holes through whicha luminous flux (luminous flux A in FIG. 7) being the phase differenceimage passes is provided. In the phase difference optical systemaccording to the present embodiment, a luminous flux (luminous flux B inFIG. 7) corresponding to the ghosting light is separated from theluminous flux (luminous flux A) being the phase difference image byincreasing the thickness t of the light beam branching element 31. Whenthe luminous flux corresponding to the ghosting light does not passthrough the through hole by separating the luminous flux correspondingto the ghosting light from the luminous flux being the phase differenceimage, it is possible to prevent the ghosting light from being imaged onthe imaging element 35.

Here, as shown in FIG. 7, a luminous flux diameter of each of theluminous flux A and the luminous flux B in a position where the two eyelens filter 33 is provided is represented as φa, a diameter (that is,diameter of the diaphragm) of the through hole provided in the two eyelens filter 33 is represented as φb, and a center distance between thethrough holes is represented as d. When the optical system is adjustedin such a manner that a center of the luminous flux A is a center of thetwo eye lens filter 33, a separation distance x between the luminousflux A and the luminous flux B may satisfy a relationship shown in thefollowing inequality 101 so that the luminous flux corresponding to theghosting light does not pass through the through hole. In addition, athreshold value of the distance x calculated based on the followinginequality 101 may be corrected to a larger value in consideration ofdefocus characteristics, and the like of the optical system.

$\begin{matrix}{x > \frac{\left( {{\varphi \; a} + {\varphi \; b} + d} \right)}{2}} & \left\lbrack {{Inequality}\mspace{14mu} 101} \right\rbrack\end{matrix}$

In addition, as described above, the separation distance x is comparedwith the thickness t of the light beam branching element 31, aproportional constant thereof is a unique one in the optical system.Accordingly, the thickness t of the light beam branching element 31 maybe determined so as to satisfy a relationship shown in the followinginequality 102. Here, in the following inequality 102, a coefficient kis an inverse number of the unique proportional constant in the opticalsystem between the separation distance x and the thickness t of thelight beam branching element 31.

$\begin{matrix}{t > {k \times \frac{\left( {{\varphi \; a} + {\varphi \; b} + d} \right)}{2}}} & \left\lbrack {{Inequality}\mspace{14mu} 102} \right\rbrack\end{matrix}$

As described above, in the defocus quantity detection unit 30 accordingto the present embodiment, the thickness t of the light beam branchingelement 31 is at least a predetermined threshold value, so that theghosting light generated by the light beam branching element 31 may besufficiently separated from the reflected light used in the process inthe microscope 1. The separated ghosting light as described above isshielded by the two eye lens filter provided within the phase differenceoptical system, so that the separated ghosting light is not imaged onthe imaging element 35 within the optical system. Thus, in themicroscope 1 according to the present embodiment, it is possible toeliminate the ghosting light generated by the light beam branchingelement 31.

In addition, in the imaging element 24 provided on the transmitted sideof the light beam branching element 31, it is possible to suppresseffects due to the ghosting light to be not higher than the noise levelof the imaging element 24 by adjusting the coating, and the likeperformed on the light beam branching element 31. Thus, even in theimaging element provided on the transmitted side of the light beambranching element 31, it is possible to eliminate the effects due to theghosting light.

Second Embodiment

The microscope 1 according to the first embodiment installs the phasedifference optical system on the reflected light side of the light beambranching element 31, and installs the imaging element 24 of themagnified image imaging unit 20 on the transmitted light side of thelight beam branching element 31. In the microscope 1 according to asecond embodiment, which will be described below, the phase differenceoptical system is installed on the transmitted light side of the lightbeam branching element 31, and the imaging element 24 of the magnifiedimage imaging unit 20 is installed on the reflected light side of thelight beam branching element 31.

In the microscope 1 according to the present embodiment, both thetransmitted light and the reflected light branched by the light beambranching element 31 are used in the process in the microscope 1. Thefront surface reflected light is used with respect to the reflectedlight reflected from the light beam branching element 31, so that it ispossible to suppress effects due to the plate thickness of the lightbeam branching element 31, however, the transmitted light transmittedthrough the light beam branching element 31 is influenced by the effectsdue to the plate thickness of the light beam branching element 31.

In addition, in order to branch the light beam transmitted through thesample, the light beam branching element 31 is installed to have aninclination with respect to the normal direction of the incident lightbeam, as shown in FIG. 1. Due to this, an amount of deviation of thelight beam due to the image height is changed, so that there arepossibilities that distortion, a chromatic aberration of magnification,a wavefront aberration, and the like are changed.

For example, it is assumed that the light beam transmitted through thecondenser lens 23 is branched by the light beam branching element 31,and the branched light beam is detected in the imaging element 24 of themagnified image imaging unit 20 or the defocus quantity detection unit30 (phase difference optical system). In this case, in the phasedifference optical system, the light beam is detected in the two eyelens 34, and the light beam is injured in the diaphragm (two eye lensfilter), so that a numerical aperture of an incident side (NA) becomessmaller than that in a case where the light beam is detected in theimaging element 24.

FIG. 8 is a graph showing a relationship between a size of a wavefrontaberration (WFE) in light propagated in the imaging system (magnifiedimage imaging unit 20) and light propagated in the imaging system andthe phase difference optical system, and the plate thickness of thelight beam branching element 31. As shown in FIG. 8, since the numericalaperture of the incident side becomes smaller, it is found that effectsof the wavefront aberration due to the plate thickness is reduced in thephase difference optical system.

Thus, as shown in FIG. 9, in the microscope 1 according to the presentembodiment, the defocus quantity detection unit 30 (phase differenceoptical system) is installed on the transmitted light side of the lightbeam branching element 31, and the magnified image imaging unit 20 (thatis, the imaging optical system) is installed on the reflected light sideof the light beam branching element 31.

Since the imaging element 24 included in the magnified image imagingunit 20 is an imaging element used for precisely imaging the magnifiedimage of the sample, the imaging element 24 is sensitive to the effectsof aberration. Accordingly, the optical system of the magnified imageimaging unit 20 is provided on the reflected light side of the lightbeam branching element 31, and only the front surface reflected light ofthe light beam branching element 31 is used, so that it is possible toeliminate the effects of aberration caused by the thickness of the lightbeam branching element. Further, when the magnified image imaging unit20 is provided, it is preferable to reflect the sample transmitted lightfrom the stage side of the light beam branching element 31.

In addition, there are possibilities that the transmitted light side ofthe light beam branching element 31 is influenced by the effects ofaberration such as the distortion, the chromatic aberration ofmagnification, the wavefront aberration, and the like, however, thephase difference optical system is provided on the transmitted lightside of the light beam branching element 31 to thereby reduce theeffects due to aberration, as shown in FIG. 8.

Further, the light beam branching element 31 according to the presentembodiment that is the same as that according to the first embodimentmay be used, except that the front surface transmittance T1<0.11, andthe front surface reflectance R1>0.89 are satisfied. The above describedlight beam branching element 31 is adopted, so that it is possible toeliminate the ghosting light, which is propagated in the phasedifference optical system, using the two eye lens filter 33, and tosuppress an intensity of the ghosting light imaged to the imagingelement 24 so as not to affect the process of the imaging element 24.

In addition, the condenser lens 32, the two eye lens filter 33, the twoeye lens 34, and the imaging element 35 according to the presentembodiment, which are the same as those according to the firstembodiment, may be used. Thus, the repeated description thereof will beherein omitted.

As described above, in the microscope 1 according to the presentembodiment, the phase difference optical system is installed on thetransmitted light side of the light beam branching element 31, and theimaging element 24 of the magnified image imaging unit 20 is installedon the reflected light side of the light beam branching element 31, sothat it is possible to eliminate the ghosting caused by the light beambranching element, and to eliminate effects of various aberrations inthe imaging element 24. As a result, it is possible to further improveaccuracy of the microscope image imaged in the imaging element 24.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope and without diminishing itsintended advantages. It is therefore intended that such changes andmodifications be covered by the appended claims.

The application is claimed as follows:
 1. A microscope, comprising: afirst imaging optical system that images sample transmitted lighttransmitted through a sample provided on a stage; and a second imagingoptical system that images a part of the sample transmitted lightbranched from the first imaging optical system, wherein the secondimaging optical system includes a light beam branching element thatbranches the part of the sample transmitted light from the first imagingoptical system, and has a thickness of a predetermined threshold ormore, an imaging element that images a phase difference image of thebranched sample transmitted light, one or a plurality of opticalelements that images an image of the phase difference image of thebranched sample transmitted light on the imaging element, and a filterthat shields a part of the branched sample transmitted light imaged onthe imaging element.
 2. The microscope according to claim 1, wherein thesample transmitted light reflected by the light beam branching elementis imaged in the first imaging optical system, and the phase differenceimage of the sample transmitted light transmitted through the light beambranching element is imaged in the second imaging optical system.
 3. Themicroscope according to claim 1, wherein the sample transmitted lighttransmitted through the light beam branching element is imaged in thefirst imaging optical system, and the phase difference image of thesample transmitted light reflected by the light beam branching elementis imaged in the second imaging optical system.
 4. The microscopeaccording to claim 2, wherein the filter is a diaphragm in which athrough hole set for allowing a luminous flux set which becomes thephase difference image to pass therethrough is provided, and thethickness of the light beam branching element has a larger value than afeature value calculated based on a diameter of the through hole, acenter distance between the through holes of the through hole set, and aluminous flux diameter at a position of the filter of the sampletransmitted light.
 5. The microscope according to claim 4, wherein thethickness of the light beam branching element has a larger value than afeature value calculated based on the following Inequality 1, which isrepresented as $\begin{matrix}{t > {k \times \frac{\left( {{\varphi \; a} + {\varphi \; b} + d} \right)}{2}}} & {{Math}.\mspace{14mu} 1}\end{matrix}$ where t denotes a thickness of the light beam branchingelement, k denotes a specific constant in an optical system, φa denotesa luminous flux diameter at a filter position of the sample transmittedlight, φb denotes a diameter of the through hole, and d denotes a centerdistance of through holes of the through hole set.
 6. A ghostingelimination method, comprising: branching, by a light beam branchingelement having a thickness of a predetermined threshold or more, a partof sample transmitted light transmitted through a sample provided on astage; and shielding, by an imaging element for imaging a phasedifference image of the branched sample transmitted light and a filterprovided between the imaging element and the light beam branchingelement, ghosting light caused by a corresponding light beam branchingelement from the part of the sample transmitted light branched by thelight beam branching element.